Estrogens for treating ALS

ABSTRACT

A method for preventing and treating ALS by administering a phytoestrogen, preferably genistein.

RELATED APPLICATION

This application is a § 1.53(b) continuation of U.S. Ser. No.09/455,846, filed Dec. 7, 1999, now U.S. Pat. No. 6,334,998 whichapplication is expressly incorporated by reference herein.

TECHNICAL FIELD

The invention relates to the treatment and prevention of amyotrophiclateral sclerosis (ALS) by the administration of estrogen. Pretreatmentwith an estrogen compound, for example, with the estrogenic isoflavone,genistein, prevents pathologic conditions associated with ALS and delaysdisease onset in individuals at high risk for ALS.

BACKGROUND

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease,is a progressive, fatal neurodegenerative disorder involving the motorneurons of the cortex, brain stem, and spinal cord (Hirano, A., 1996,Neurology 47 (Suppl. 2), S63-S66). It is a degenerative disease of upperand lower motor neurons that produces progressive weakness of voluntarymuscles, with eventual death. The onset of disease is usually in thefourth or fifth decade of life, and affected individuals succumb within2 to 5 years of disease onset. ALS occurs in both sporadic and familialforms. About 10% of all ALS patients are familial cases, of which 20%have mutations in the superoxide dismutase 1 (SOD1) gene (formerly knownas Cu,Zn-SOD), suggesting that an abnormally functioning Cu,Zn-SODenzyme may play a pivotal role in the pathogenesis and progression offamilial amyotrophic lateral sclerosis (FALS) (Rosen et al., 1993,Nature 362: 59; Siddique et al., 1991, N. Engl. J. Med. 324:1381).

More than 50 point mutations of the human SOD1 gene have been found inpatients with FALS. Most of the mutations occur at regions involved inthe subunit folding exposing the active site to the outside, leading toincreased hydroxyl radical generation. It is believed that the increasedgeneration of oxygen free radicals, especially hydroxyl radicals, bymutant SOD1, to be the initiating factor that results in the sequence ofevents leading to motor neuron death in FALS. This hypothesis issupported by recent reports that transfection of neuronal precursorcells with mutant SOD1 results in increased production of hydroxylradicals and enhanced rate of cell death by apoptosis (Liu et al., 1999,Radiat. Res. 151:133).

The incidence of ALS in males is higher than in females (Reed and Brody,1975, Am. J. Epidemiol. 101:287; Yoshida et al., 1986, Neuroepidemiology5:61), indicating a sexual dimorphic etiology. It is known that estrogentherapy can produce beneficial effects for a variety of diseases,however, estrogen has not previously been suggested as useful in thetreatment of ALS.

Estrogen treatment reportedly lowers the incidence of cardiovasculardiseases and stroke mortality 70% and 36%, respectively (Stampfer etal., 1991, N. Engl. J. Med., 325: 756-762; Paganini-Hill et al., 1988,Br Med J, 297: 519-522). Physiological events, such as improvedcerebrovascular blood flow, as well as cellular events, such asattenuation of neuronal cell death from glutamate toxicity and oxidativestress, are all involved in the neuroprotective activity of estrogen.

Phytoestrogens are non-steroidal plant compounds that have estrogenicactivity in humans and animals. Genistein is a phytoestrogen exhibitingweak estrogen activity both in vitro and in vivo (Santel et al, 1997, J.Nutr, 127:263-269; Hilakivi-Clarke et al., 1998, Oncol Rep, 5: 609-616;Zava et al., 1997, Environ Health Perspect, 105(Suppl. 3): 637-645). Invitro, genistein binds to the estrogen receptor and inducesestrogen-regulated end products (Zava et al., 1997, supra). However,genistein, has also been shown to exhibit pleiotropic biologicactivities including antioxidant activity (Ruiz-Larrea et.al., 1997 FreeRad. Res. 26:63); inhibition of tyrosine kinase activity (Uckun et.al.,1995, Science 267:886) and protective activity against radiation inducedapoptotic cell death (Uckun et.al., 1992, PNAS USA 89:9005).

Despite having been studied for over 100 years, the etiology of ALS isstill largely unknown with no clearly effective treatment or method ofprevention. Methods and compositions that prevent or ameliorate theneurologic damage indicative of ALS are needed. Accordingly, there is aneed for a method or composition that prevents or delays onset ofpathologic conditions related to ALS. In the present invention,phytoestrogens such as the isoflavone genistein, are demonstrated to beeffective as a neuroprotective compounds which can delay onset of ALSand pathological symptoms associated with this disease.

SUMMARY

The present invention provides methods for preventing and/or delayingsymptoms, or treating symptoms relating to amyotrophic lateral sclerosis(ALS). More particularly, the invention provides estrogens, particularlyphytoestrogens, and preferably estrogenic flavonoid compounds having oneor more hydroxylated aromatic rings, for the treatment of ALS. Preferredestrogens include genistein and effective analogs of genistein,coumestrol, zeanoloval,4-(4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (P131) and2,4,4′-trihydroxy deoxybenzoin (DDE-17) for administration to a subjectfor the prevention and/or treatment of ALS

Preferably, the estrogen is administered to a patient before the onsetof ALS. Administration of the estrogen to the patient, can be, forexample, systemically, or locally into a tissue.

A patient to be treated by the method of the invention is one determinedto be susceptible to ALS, one exhibiting a symptom associated with ALS,or one diagnosed as suffering from ALS. Patients susceptible to ALSinclude patients at risk of developing ALS. These would include, forexample, patients with a family history or predisposition to developingALS, and the like. Indications that a patient is susceptible to ALSinclude those individuals testing positive for molecular markersindicative of or associated with ALS. Such markers include, for example,any one of the known mutations in the SOD1 gene (Deng et al., 1993,Science, 261: 1047-1051).

Accordingly, one embodiment of the present invention includes a methodfor preventing onset of ALS, including the step of administering to asubject, prior to the onset of ALS, an effective amount of an estrogen,such as coumestrol, zearolonal, or genistein. Preferred is theadministration of a hydroxy-substituted estrogenic isoflavone having thegeneral formula I shown below. The invention also includes prevention ofdisease progression by administering such a compound; and also use ofestrogens for the treatment of ALS disease symptoms.

R is NH₂, halo, C₁-C₆ alkyl, or OR′ and n is 1 to 4, wherein at leastone R is OR′, and R′ is H, C₁-C₆ alkyl, or a sugar moiety. (It is commonfor a sugar moiety to bind the flavone via reaction with the flavone'shydroxyl groups.) One or both of the rings, A and B, each comprise oneor more hydroxyl groups (R). Preferred are phytoestrogens and theiractive estrogenic derivatives. Most preferred is the estrogenicisoflavone, genistein, shown below.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The Figures and the detailed description which follow moreparticularly exemplify these embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing delayed disease onset and mortality in maleFALS mice treated with genistein.

FIG. 2 is a graph showing delayed onset and mortality in male micetreated with the estrogens P131 and DDE-17.

FIG. 3 is an infrared (IR) spectrum of the compound DDE-17.

DETAILED DESCRIPTION

The invention provides methods for preventing, treating and/orameliorating the effects of neurological injury induced in tissues dueto ALS.

While the invention is amenable to various modifications and alternativeforms, specifics of the invention are shown by example and will bedescribed in detail. It should be understood, however, that theintention is not to limit the invention to the particular embodimentsdescribed. On the contrary, the intention is to cover all modifications,equivalents, and alternatives falling within the spirit and scope of theinvention.

Definitions

All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. As used inthis application, the following words or phrases have the meaningsspecified.

As used herein, “therapeutically effective amount” is meant to define adose or predetermined amount of the drug that provides protectionagainst damage due to ALS, or that reduces, alleviates, prevents theonset of, or prevents the progression of one or more symptoms associatedwith ALS. A amount of phytoestrogen expected to be therapeuticallyeffective is a daily dose in the range of about 10 to 100 mg/kg bodyweight.

As used herein, “alkyl”, which includes the alkyl group of “alkoxy”substituents, is meant to define both branched and straight-chainsaturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms, for example C₁-C₆. As a preferred embodiment, “alkyl”means chains of 1 to 4 carbon atoms, for example, methyl, ethyl, propyl,isopropyl, butyl, secondary butyl, t-butyl, and the like.

As used herein, “halogen” or “halo” substituent means fluoro, chloro,bromo, and iodo.

As used herein, “treating” means the prevention or reduction of severityof symptoms or effect of a pathological condition, including prolonginglife expectancy. In the context of ALS therapy, treatment includes“prevention” or inhibition of the onset of disease symptoms associatedwith or indicative of ALS, such as limb paralysis, muscle weakness,and/or neurodegeneration of the motor cortex.

As used herein, “at risk” means an individual who has a familial orgenetic predisposition indicative of ALS; an individual who carries amarker found to be associated with or correlative to ALS.

Compounds of the Invention

Compounds useful in the method of the invention are estrogens, asdescribed above. The estrogenic activity of a compound may be determinedby known methods, for example by assay of estrogen receptor bindingactivity or by bioassay of estrogenic function.

Preferred phytoestrogen compounds of the invention include genistein,coumestrol, zearolonal, Luteolin, diadzin, aminogenistein, quercetin,and the like. Most preferred are estrogenic isoflavones such asgenistein and 2,4,4′-trihydroxy deoxybenzoin (DDE-17).

The general structure of the preferred estrogens is shown below, where Ris NH₂, halo, C₁-C₆ alkyl, or OR′ and n is preferably 1 to 4, where atleast one R is OR′, and R′ is H, C₁-C₆ alkyl, or a sugar moiety.

A most preferred isoflavone is genistein, having the structure shownbelow:

Active analogs may also be used. For example, for purposes of thisinvention, an active analog of genistein is defined as a compoundstructurally related to genistein, for example a different flavonoid,which is effective in reducing the size of induced ischemic lesions intissue, for example, as analyzed in the non-invasive murine mouse modeldescribed in the Examples below. Such active analogs of genisteininclude, for example, amino-genistein and other such derivatives ofgenistein which retain estrogenic activity and the ability to amelioratesymptoms of ALS or prevent onset of ALS. It is understood that theestrogenic compounds of the invention may be modified in numerous wayswithout affecting the estrogenic activity useful in the invention. Suchmodifications, analogs, and derivatives are within the scope of“estrogens” of the invention.

The naturally occurring isoflavone, genistein, is demonstrated herein tohave protective effects against symptoms of ALS and is useful as aprophylactic agent for prevention and treatment of ALS. While not meantto limit the invention, it is believed that this protective effect isdue to the estrogenic activity of the administered phytoestrogen.

Genistein is a phytoestrogen found in soy products (Narasimhachari etal., 1953, J. Sci. Ind. Res. 12:287). Genistein may be synthesized asdescribed by Wahala et al., 1991, Journal of the Chemical Society ofPerk Trans 1:3005, and as described in the synthetic scheme shown below.

Preferred compounds of the invention are genistein and active analogsthereof. Other estrogens can also be used in the method of theinvention. These include, for example, coumesterol, zearalonol, and2,4,4′-trihydroxy deoxybenzoin (DDE-17).

Methods and Pharmaceutical Compositions

In the method of the invention, estrogens are prepared in pharmaceuticalcompositions for treatment and prevention of conditions that give riseto ALS and to neuronal tissue damage due to ALS. Patient treatment usingthe method of the present invention involves administering therapeuticamounts of an estrogen composition.

In the context of the present invention, the terms “treat”, “therapy”,and the like are meant to include methods to alleviate, slow theprogression, prevent, attenuate, or cure the treated disease.

The pharmaceutical compositions of the present invention include anestrogen in effective unit dosage form and a pharmaceutically acceptablecarrier. As used herein, the term “effective amount”, “effective unitdosage” or “effective unit dose” is meant to define a predeterminedamount of the drug that provides protection of cells or tissues againstdamage due to onset or progression of ALS, or lessons a symptomindicative of ALS such as limbic paralysis, muscle wasting, weakness orcramping. An amount of isoflavone expected to be effective is a dailydose in the range of about 10 to 100 mg/kg.

Pharmaceutically acceptable carriers are materials useful for thepurpose of administering the medicament, which are preferably non-toxic,and can be solid, liquid, or gaseous materials, which are otherwiseinert and medically acceptable and are compatible with the activeingredients. Suitable pharmaceutical carriers and their formulations aredescribed in Martin, “Remington's Pharmaceutical Sciences,” 15th Ed.;Mack Publishing Co., Easton (1975); see, e.g., pp. 1405-1412 and pp.1461-1487. Such compositions will, in general, contain an effectiveamount of the active compound together with a suitable amount of carrierso as to prepare the proper dosage form for proper administration to thehost.

The pharmaceutical composition may also be combined with or conjugatedto specific delivery agents, including targeting antibodies and/orcytokines. The pharmaceutical composition can include pharmaceuticallyacceptable salts of the active estrogen. Pharmaceutically acceptablesalts include salts of any potentially charged group in the compound,such as a salt of any amine. Suitable salts include gluconate salts,chloride salts, and the like. Such conjugated estrogens, salts,prodrugs, and the like are considered within the scope of “estrogens” ofthe invention.

Conjugation to a Targeting Moiety

The composition of the invention can be targeted for specific deliveryto the cells to be treated by conjugation of the compounds to atargeting moiety. Targeting moiety useful for conjugation to thecompounds of the invention include antibodies, cytokines, and receptorligands expressed on the cells to be treated.

The term “conjugate” means a complex formed with two or more compounds.

The phrase “targeting moiety” means a compounds which serves to deliverthe compound of the invention to a specific site for the desiredactivity. Targeting moieties include, for example, molecules whichspecifically bind molecules present on a cell surface. Such targetingmoieties useful in the invention include anti-cell surface antigenantibodies. Particularly useful targeting moieties for targeting thecompounds of the invention to cells for therapeutic activity includethose ligands that bind known antigens or receptors present on theneurons to be treated.

Administration Methods

A composition of the invention may be formulated with conventionalpharmaceutically acceptable parental vehicles for administration byinjection. These vehicles comprise substances which are essentiallynontoxic and nontherapeutic such as water, saline, Ringer's solution,dextrose solution, Hank's solution, a perfusion solution, and the like.It is to be understood that isoflavone formulations may also includesmall amounts of adjuvants such as buffers and preservatives to maintainisotonicity, physiological and pH stability. The compositions accordingto the invention can be presented in unit dose form in ampules or inmulti-dose containers with an added preservative. The compositions cantake such forms as suspensions, solutions, or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing, and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile, pyrogen-free buffer saline, before use. Thepresent compositions can also be in the form of encapsulated liposomesor other sustained release formulations.

As indicated by the above formulation, the composition can beadministered parenterally. The composition can be administered by knowntechniques such as orally, for example, in tablets, capsules,enterically coated capsules and the like, or in drops, among othermethods such as by inhalation spray, topically, by transdermal patchesor by absorption through a mucous membrane, or rectally, in dosage unitformulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants or vehicles. When administeredintravenously, it can be delivered as a bolus or on a continuous basis.The composition can be administered intrarterially such as through acatheter. A preferred method of administration is intravenousadministration of a sustained release formulation.

For oral administration as a suspension, the compositions can beprepared according to techniques well-known in the art of pharmaceuticalformulation. The compositions can contain microcrystalline cellulose forimparting bulk, alginic acid or sodium alginate as a suspending agent,methylcellulose as a viscosity enhancer, and sweeteners or flavoringagents. As immediate release tablets, the compositions can containmicrocrystalline cellulose, starch, magnesium stearate and lactose orother excipients, binders, extenders, disintegrants, diluents andlubricants known in the art.

For administration by inhalation or aerosol, the compositions can beprepared according to techniques well-known in the art of pharmaceuticalformulation. The compositions can be prepared as solutions in saline,using benzyl alcohol or other suitable preservatives, absorptionpromoters to enhance bioavailability, fluorocarbons or othersolubilizing or dispersing agents known in the art.

For rectal administration as suppositories, the compositions can beprepared by mixing with a suitable non-irritating excipient, such ascocoa butter, synthetic glyceride esters or polyethylene glycols, whichare solid at ambient temperatures, but liquify or dissolve in the rectalcavity to release the drug.

The dose of the estrogen formulation to be administered will depend uponthe patient and the patient's medical history, and the severity of thedisease process. However, the dose should be sufficient to effectivelyprecondition against ALS, or to effectively prevent, reduce, or inhibitdamage due to ALS. Dosages for adult humans envisioned by the presentinvention and considered to be therapeutically effective will preferablyrange from between about 10 mg/kg and about 100 mg/kg. These doses maybe repeated up to several times per day. In addition, lower and higherdoses may be more appropriate depending on the individual patient andthe degree of condition to be treated.

Typically, the compositions of the invention will be administered to apatient (human or other animal, including mammals) in need thereof, inan effective amount to reduce or prevent damage from ALS. The presentcompositions can be given either orally, intravenously, intramuscularlyor topically.

The composition can be administered to a patient or locally to a tissue.When administered to a patient, the composition can be administeredsystemically, or directly to an organ or tissue, for example through acatheter to the blood stream entering the tissue or organ, or via thespinal column for access to cerebral fluids of the brain. Alternatively,the composition can be administered via injection directly into thetissue or organ, or into other fluids or solutions that enter the organ,such as lymph or iv solutions.

The composition is preferably administered before damage due to ALSoccurs. For example, if it is known that a patient is at high risk ofALS because of diagnostic indicators, or a family history of ALS,prophylactic treatment with an isoflavone in accord with the inventionis indicated. Preferably, the estrogen is administered prior to patientpresentation of symptoms indicative of ALS.

Clinical Features of ALS

ALS generally presents with a characteristic pattern of lesionsinvolving motor neurons of the brain stem and anterior horn area of thespinal cord. There is also involvement of the large pyramidal neurons ofthe motor cortex, with pathological changes found in the extramotorcortex, in the cerebrospinal and sensory systems as well as in thesubcortical regions (Hughes, J. T., 1982, Pathology of amyotrophiclateral sclerosis. In: Rowland LP (ed) Human motor neuron disease.(Advances in Neurology, vol 36). The criteria for diagnosis of ALS hasbeen established by the World Federation of Neurology (Brooks, B. R.,1994, El Escorial World Federation of Neurology criteria for thediagnosis of amyotyrophic lateral sclerosis. Subcomittee on MotorNeurone Diseases/Amyotrophic Lateral Sclerosis of the World Federationof Neurology Research Group on Neuromuscular Diseases and the ElEscorial ‘Clinical limits of amyotrophic lateral sclerosis’ workshopcontributors. J. Neurol Sci [Suppl] 124:96-107). An overview of theneuropathologic findings in patients with ALS is presented by A. Hirano(1996, Neuropathology of ALS: an overview, Neurology, 47 (Suppl 2):S63-S66).

ALS is characterized by paralysis, muscular atrophy, spasticity, and avariety of other motor signs. The natural history of ALS is welldocumented (Munset T. L., 1992, The natural history of amyotrophiclateral sclerosis. In: Handbook of Amyotrophic Lateral Sclerosis, SmithR A (eds.), Chapter 2, pp. 39-63, Marcel Dekker, Inc.: New York). Thepresenting symptoms of ALS include, for example, muscle wasting orweakness of the hands or legs. Occasionally, cramps and fasciculationsprecede the common presenting symptoms. Bulbar symptoms consisting ofdysarthria or dysphagia appear as the disease progresses, but can alsobe the presenting complaints in some of the patients. Such patients maybe placed on a prophylactic regimen as long as risk remains high asdetermined by standard diagnostic indicators. The preferred method is toadminister an effective amount of a phytoestrogenic compound, such as anisoflavone to a patient at high risk for ALS.

A patient or tissue may also be treated by the method of the inventionafter some damage due to ALS has occurred to minimize further damagefrom additional neuropathological events. An isoflavone according to amethod of the invention can be administered for example after atransient episode. An effective amount is that amount which reducessymptoms such as lymbic paralysis.

In general, a dose which delivers about 10 to 100 mg/kg body weightadministered for at least about 24 hours after presentation or diagnosisis expected to be effective, although more or less may be useful. Oneskilled in the art will use standard procedures and patient analysis tocalculate the appropriate dose, extrapolating from the data provided inthe Examples. In addition, the compositions of the invention may beadministered in combination with other therapies for ALS. In suchcombination therapy, the administered dose of the isoflavone may be lessthan for single drug therapy.

A patient presenting with symptoms indicative of ALS may also be treatedby the method of the invention to prevent progression of the disease orto prevent the development of more severe symptoms. A patient with ALSmay also be treated by the method of the invention to lessen thesymptoms of ALS and/or to extend the patient's longevity. As disclosedbelow, genistein inhibits disease onset and delays death in an animalmodel of ALS, thus providing an effective method for the treatment andprevention of ALS.

One or more of the following clinical evaluations can be used to assessprogress/prevention of ALS disease.

(1) Quantitative Strength and Functional markers: The TUFTS QuantitativeNeuromuscular Examination (TQNE) is a well standardized, reliable,validated test to measure strength and function in ALS. The testinvolves measurement of maximum voluntary isometric contraction (MVIC)of 8 muscle groups in the arms using a strain gauge tensiometer. Thismeasurement is a standard for clinical trials in ALS.

(2) Functional measures: The ALS Functional Rating Scale (ALSFRS) is aneasily administered ordinal rating scale used to determinepatients'assessment of their ability and independence in 10 functionalactivities. Validity has been established by correlating ALSFRS scoreswith change in strength over time. The ALSFRS is generally a secondaryoutcome measure in clinical trials.

(3) Measures of Upper Motor Neuron Function by traditional methods.

EXAMPLES

The present invention may be better understood with reference to thefollowing examples. These examples are intended to be representative ofspecific embodiments of the invention, and are not intended as limitingthe scope of the invention.

Example 1 Sexual Dimorphism in a Murine Model of FALS

Heterozygous transgenic mice carrying the human SOD-1 (G93A) wereobtained from Jackson Laboratory (Bar Harbor, Me., USA). These mice weremonitored everyday for limbic paralysis and death. Onset of FALS wasdefined as the first day upon which limbic paralysis was observed.Life-table analysis was performed using Statview, Abacus Concepts, Inc.(Berkeley, Calif.).

There are currently several transgenic animal models of ALS (Borchelt etal., 1998, Transgenic mouse models of Alzheimer's disease andamyotrophic lateral sclerosis, Brain Pathology, 8: 735-757). The G93AHuSOD1 is a well established animal model of familial ALS which has beendescribed previously in detail (Dal Canto M C, Gurney Me., 1994, Thedevelopment of central nervous system pathology in a murine transgenicmodel of human amyotrophic lateral sclerosis. Am J. Pathol 145: 1-9;Gurney et al., 1994, Motor neuron degeneration in mice that express ahuman Cu,Zn superoxide dismutase mutation, Science 264:1772-1775).Levels of SOD1 activity are elevated to about five times above that ofendogenous SOD 1 in these mice. In this model, the mice develop hindlimb weakness, a coarse coat, poor grooming, and reduced stride lengthat about 3 months of age. By five months of age, a majority of the miceare moribund.

Among individuals diagnosed with ALS, there is a high male to femaleratio of incidence (Reed, D. M., Brody J. A., 1975, Am J. Epidemiol.101:287-301; Yoshida et al., 1986, Neuroepidemiology 5:61-70). Theabove-described G93A HuSOD1 mice were employed to examine the sexualdimorphism of ALS in an animal model of this disease.

As shown in FIG. 1, male FALS mice (open circles) exhibited an earlierdisease onset and death as compared to female FALS mice (triangles).Female FALS mice exhibited significantly delayed disease onset andmortality when compared to male FALS mice (p=0.001 and p=0.01,respectively, log-rank statistic). These results indicate that thefemale sex hormone, estrogen, may be protective against FALS.

Example 2 Genistein is Neuroprotective Against ALS

The ability of the isoflavone genistein to protect against neuraldegeneration in a murine model of familial ALS was evaluated. Genisteinwas synthesized as reported previously (Uckun et al., 1995, Science,267: 886-891).

Heterozygous transgenic mice carrying the human SOD-1 (G93A) wereobtained from Jackson Laboratory (Bar Harbor, Me., USA). The mice weretreated either with vehicle or genistein at 16 mg/kg twice a day,everyday, except on weekends. The mice were monitored everyday for limbparalysis and death. Onset of FALS was defined as the first day uponwhich limbic paralysis was observed. Life-table analysis was performedusing Statview, Abacus Concepts, Inc. (Berkeley, Calif.).

FALS mice were divided into the 4 groups: (males treated with vehicle(open circles, n=13) or genistein (filled circles, n=13); and femalestreated with vehicle (triangles, n=15)or genistein (squares, n=13)).Genistein treatment delayed the disease onset and mortality in male FALSmice (p=0.02 and p=0.02, respectively, long-rank statistic), with curvessuperimposible on those of female FALS mice (see FIG. 1), indicatingthat the phytoestrogen, genistein, is neuroprotective in FALS mice.

Phytoestrogen treated females (squares) did not exhibit a delay indisease onset in comparison to untreated females (triangles), suggestingthat phytoestrogens and endogenous estrogen operate through commonpathway(s). Taken together, these results provide unprecedented evidencethat phytoestrogens, and particularly genistein, are useful as aprophylactic agents for prevention of neurodegenerative symptoms inindividuals who are at high risk for ALS as well as providing effectivetreatment against ALS.

Example 3 Compounds for Neuroprotection Against ALS

The ability of 4-(4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline(P131) and 2,4,4′-trihydroxy deoxybenzoin (DDE-17) to protect againstneural degeneration in familial ALS was evaluated using the methodsdescribed above for Example 2. P131 and DDE17 have the structures shownbelow:

DDE-17 was synthesized and characterized as described below in Example4. The synthesis and characterization of P131 was previously describedin published PCT application no. PCT/US99/11767.

As shown in FIG. 2, male mice treated with P131 (n=25, filled squares)and particularly those treated with DDE-17 (filled circles, n=13)demonstrated delayed onset and mortality as compared with thevehicle-treated control (open circles, n=28).

Example 4 Synthesis and Characterization of DDE-17

2, 4, 4′-trihydroxy deoxybenzoin (DDE-17) was produced according to thefollowing scheme:

Trihydroxy benzine (25m Mole) and 4-hydroxy phenyl acetic acid (25mMole) was transferred to a flask dried under nitrogen atmosphere. BF₃Et₂O (25 ml) was added and the mixture allowed to reflux on a steam bathat 70-80° C. Dry DMF was added under N₂ atmosphere.

An aliquot was poured into ice water acidified with HCl, extracted withCH₂Cl₂, and dried over NaSO₄. Solvent was removed under vacuum and IRtaken, dissolved in CHCl. The reulting IR data is shown in FIG. 3: IR4745, 3960, 2923, 1708, 1631, 1596 cm.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

All publications and patent applications in this specification areindicative of the level of ordinary skill in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated by reference.

We claim:
 1. A method for treating or delaying the onset of ALS,comprising: administering 4-(4′-hydroxyphenyl)amino-6,7-dimethoxyquinazoline to a patient suffering from or at risk ofdeveloping ALS in order to reduce symptoms or delay onset of ALS.
 2. Amethod for ameliorating neuronal tissue damage in an ALS patientcomprising administering an effective amount of4-(4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline to a patientdiagnosed as having ALS.